Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses - PubMed (original) (raw)
Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses
Shin Murakami et al. J Virol. 2017.
Abstract
Akabane virus (AKAV) and Schmallenberg virus (SBV) are members of the genus Orthobunyavirus, which are transmitted by arthropod vectors with a broad cellular tropism in vitro as well as in vivo Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system and measured the quantities of binding of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least in vitro, to promote virus replication in susceptible cells.IMPORTANCE AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines.
Keywords: Akabane virus; Schmallenberg virus; cell entry; heparan sulfate.
Copyright © 2017 American Society for Microbiology.
Figures
FIG 1
Effects of heparin and heparinase treatment on AKAV and SBV infections. (A) Effects of heparin on AKAV and SBV plaque reduction. Various concentrations of heparin were incubated for 30 min at room temperature with 100 PFU of AKAV(OBE-1), AKAV(Iriki), or SBV, and the ability of heparin to reduce plaque formation was assessed. (B) Effects of heparinase treatment on AKAV and SBV infectivity. HmLu-1 cells were treated with various concentrations of heparinase II, followed by AKAV or SBV infection. Cells were stained for AKAV or SBV antigen, and positive cells were counted under a fluorescence microscope. For VSV-ΔG-GFP-infected cells, GFP-positive cells were counted under a fluorescence microscope. Results are expressed in percentages relative to cells that were not treated with heparinase. The data are reported as the mean value with standard deviations for three independent experiments.
FIG 2
AKAV and SBV growth kinetics and infectivity in HSPG-KO HmLu-1 cells. (A) Flow cytometric analysis of EXT2-KO HmLu-1 cells. CRISPR-Cas9-mediated EXT2-KO cell clones (EXT2-1 and EXT2-2) were labeled with anti-heparan sulfate mouse monoclonal antibody (10E4) (black) or with isotype control (red) and analyzed by flow cytometry (FACSVerse; BD Biosciences). Representative data (one out of three clones of random-KO, EXT2KO-1, and EXT2KO-2) are shown. (B) Growth kinetics of AKAV or SBV in HSPG-KO cells. AKAV(OBE-1), AKAV(Iriki), or SBV was inoculated onto three clones of random-KO, EXT2KO-1, and EXT2KO-2 cells at a multiplicity of infection of 0.01. Virus titers were determined by plaque assay in normal HmLu-1 cells. The data are reported as the mean titer for three clones of each KO cell (EXT2KO-1, EXT2KO-2, or random-KO) with standard deviations. (C) Infectivities of AKAV and SBV in HSPG-KO cells. Random-KO or HSPG-KO cells were infected with AKAV(OBE-1), AKAV(Iriki), SBV, or control VSV-ΔG-GFP. Cells were stained for AKAV or SBV antigen, and positive cells were counted under a fluorescence microscope. For VSV-ΔG-GFP-infected cells, GFP-positive cells were counted under a fluorescence microscope. Results are expressed as percentages relative to the number of positive random-KO cells. The data are reported as the mean value for three clones of each KO cell (EXT2KO-1, EXT2KO-2, or random-KO) with standard deviations. (D) Infectivities of VSV pseudotyped with AKAV Gn/Gc (VSV-ΔG-GFP/AKAV) in HSPG-KO cells. Random-KO or HSPG-KO cells were infected with VSV-ΔG-GFP/AKAV or control VSV-ΔG-GFP. GFP-positive cells were counted under a fluorescence microscope. Results are represented as percentages relative to the number of positive random-KO cells. The data are shown as the mean value for three clones of each KO cell (EXT2KO-1, EXT2KO-2, or random-KO) with standard deviations.
FIG 3
AKAV and SBV binding assays in HSPG-KO cells. (A) Real-time RT-PCR for the quantification of cell surface-attached viruses. AKAV(OBE-1) or SBV was incubated with HSPG-KO cells at 4°C. After a washing step, total RNAs were extracted. AKAV or SBV S RNAs were quantified by one-step real-time RT-PCR. For relative quantification, standard curves of AKAV or SBV S RNA and GAPDH were prepared by serial dilution of a mixture of total RNA from uninfected HmLu-1 cells and RNA extracted from AKAV(OBE-1) or SBV-containing supernatants. Results are expressed as the percentages relative to the levels in random-KO cells. The results are representative of three different experiments. (B) Sandwich ELISA for the detection of N proteins of AKAV attached to cell surfaces. AKAV(OBE-1) was inoculated onto HSPG-KO or random-KO HmLu-1 cells and left for 1 h at 4°C. After a washing step, the cells were lysed, and the lysates were added to the anti-AKAV N monoclonal antibody (5E8)-coated wells of 96-well ELISA plates (Maxisorp, Nunc), followed by incubation with biotinylated anti-AKAV mouse polyclonal antibody. Subsequently, the wells were incubated with avidin-biotinylated horseradish peroxidase (HRP) complex (Vectastain ABC kit; Vector Laboratories). A 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was used for detection, and optical density values were measured. Results are expressed as percentages relative to the number of positive random-KO cells. The results are representative of three independent experiments.
FIG 4
Rescue of AKAV and SBV infectivities by adding back the EXT2 gene to EXT2-KO cells. (A) Flow cytometric analysis of adding back the EXT2 gene in EXT2-KO-1 HmLu-1 cells. Cells were labeled with anti-heparan sulfate mouse monoclonal antibody (10E4) and analyzed by flow cytometry (FACSVerse; BD Biosciences). (B) Infectivities of AKAV and SBV in cells with EXT2 added back. The cells were infected with AKAV(OBE-1), AKAV(Iriki), or SBV. Cells were stained for AKAV or SBV antigen, and positive cells were counted under a fluorescence microscope. Results are expressed as percentages relative to the number of wild-type cells. The data are reported as the mean value with standard deviations for three independent experiments.
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